A Fusion Protein Vaccine Containing OprF Epitope 8, OprI,and Type A and B Flagellins Promotes Enhanced Clearance of Nonmucoid Pseudomonas aeruginosa

Although chronic Pseudomonas aeruginosa infection is the major cause of morbidity and mortality in cystic fibrosis (CF) patients, there is no approved vaccine for human use against P. aeruginosa. The goal of this study was to establish whether a multivalent vaccine containing P. aeruginosa type A and B flagellins as well as the outer membrane proteins OprF and OprI would promote enhanced clearance of P. aeruginosa. Intramuscular immunization with flagellins and OprI (separate) or OprI-flagellin fusion proteins generated significant antiflagellin immunoglobulin G (IgG) responses. However, only the fusions of OprI with type A and type B flagellins generated OprI-specific IgG. Immunization with a combination of OprF epitope 8 (OprF_311-341), OprI, and flagellins elicited high-affinity IgG antibodies specific to flagellins, OprI, and OprF that individually promoted extensive deposition of C3 on P. aeruginosa. Although these antibodies exhibited potent antibody-dependent complement-mediated killing of nonmucoid bacteria, they were significantly less effective with mucoid isolates. Mice immunized with the OprF_311-341-OprI-flagellin fusion had a significantly lower bacterial burden three days postchallenge and cleared the infection significantly faster than control mice. In addition, mice immunized with the OprF_311-341-OprI-flagellin fusion had significantly less inflammation and lung damage throughout the infection than OprF-OprI-immunized mice. Based on our results, OprF_311-341-OprI-flagellin fusion proteins have substantial potential as components of a vaccine against nonmucoid P. aeruginosa, which appears to be the phenotype of the bacterium that initially colonizes CF patients.Cystic fibrosis (CF) is a hereditary disease that is linked to a defective CF transmembrane receptor (CFTR) (48). In CF patients, the presence of a defective CFTR protein leads to dehydrated mucosal surfaces and disruption of ion transport. In the initial stages of disease, CF patients are infected with Staphylococcus aureus and Haemophilus influenzae but eventually become infected with nonmucoid Pseudomonas aeruginosa, a gram-negative opportunistic pathogen that is the major cause of morbidity and mortality in these patients (5, 27, 28, 61). Following colonization, P. aeruginosa undergoes a mucoid conversion to an alginate-overexpressing phenotype that is associated with biofilm development and enhanced resistance to antibiotic therapy (28). CF is characterized by lung inflammation mediated, in part, by chronic P. aeruginosa infection. P. aeruginosa possesses numerous virulence factors that facilitate evasion of the immune system (15, 37, 43, 49). For example, P. aeruginosa secretes enzymes such as alkaline protease and elastase, which degrade complement components and thus limit the role of complement in the clearance of early pulmonary P. aeruginosa infections (16). The critical role of complement in the clearance of P. aeruginosa is evidenced by the observation that C3 and C5 knockout mice were unable to clear P. aeruginosa after challenge (40, 69). In addition, P. aeruginosa expresses lipopolysaccharide variants that interfere with C3b deposition (52).Initial efforts to develop a P. aeruginosa vaccine focused primarily on lipopolysaccharide. Although vaccination with P. aeruginosa lipopolysaccharide was effective in several animal models and led to the production of highly opsonic antibodies, the efficacy in human trials was limited by antigenic diversity of O antigens among P. aeruginosa isolates (11). Since flagellin, OprI, and OprF exhibit conserved amino acid sequences, more recent studies have focused on these proteins as potential vaccine antigens (14, 26, 31, 67, 68).P. aeruginosa possesses two types of flagellins, type A and type B, that differ in amino acid composition and length of the hypervariable region. P. aeruginosa flagellins have the unique property of being potent adjuvants as well as protective antigens (8, 32, 42, 50). Previous work has established flagellin as a potent adjuvant in mice (1, 3, 9, 10, 23, 33-35, 45, 53, 56) as well as cynomolgus and African green monkeys (24, 36). A phase III clinical trial of P. aeruginosa flagellins in CF patients demonstrated that the vaccine was well tolerated and caused a 30% reduction in the incidence of infection (12). In related studies, immunization with the OprI antigen of P. aeruginosa and an appropriate adjuvant elicited a protective response in mice that correlated with the titer of OprI-specific immunoglobulin G (IgG) (14). In addition, an adenovirus expressing epitope 8 (amino acids 311 to 341) of OprF (i.e., the OprF_311-341 protein) provided protection against acute P. aeruginosa infection (67, 68). Several investigators have focused on a fusion peptide containing OprF and OprI as a potential vaccine candidate. Although large amounts of this protein were required for an optimal response, immunization with an OprF-OprI fusion protein resulted in a 95-fold increase in the 50% lethal dose for mice. A subsequent study in burn patients revealed that an OprF-OprI fusion protein was immunogenic and well tolerated (26, 31).Although these experimental P. aeruginosa vaccines have shown promise in initial clinical trials, none have achieved the level of response required for protection against P. aeruginosa in CF patients. After a critical review of the literature, we have identified several features that are critical for an effective P. aeruginosa vaccine: the presence of a potent adjuvant, the ability to induce high-titer antigen-specific IgG that exhibits a high degree of functional activity (for example, complement activation), multivalency, and the ability to induce a robust memory response. To that end, we generated a multivalent vaccine containing type A and B flagellins, OprF, and OprI and have evaluated its immunogenicity and protective potential. A key feature of the vaccine is the presence of flagellin, a potent adjuvant that signals via Toll-like receptor 5 (TLR5).     

Proteomic Identification of OprL as a Seromarker for Initial Diagnosis of Pseudomonas aeruginosa Infection of Patients with Cystic Fibrosis

Identification of new immunogenic antigens that diagnose initial Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF) alone or as an adjunct to microbiology is needed. In the present study, a proteomic analysis was performed to obtain a global assessment of the host immune response during the initial P. aeruginosa infection of patients with CF. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to identify outer membrane protein L (OprL), a non-type III secretion system (TTSS) protein, as an early immunogenic protein during the initial P. aeruginosa infection of patients with CF. Longitudinal Western blot analysis of sera from 12 of 14 patients with CF detected antibodies to OprL during the initial P. aeruginosa infection. In addition, also detected were antibodies to ExoS, ExoU, or ExoS and ExoU, the latter indicating sequential P. aeruginosa infections during initial infections. Detection of serum reactivity to OprL, along with proteins of the TTSS, and in conjunction with microbiology may diagnose initial P. aeruginosa infections in patients with CF.Cystic fibrosis (CF) is an autosomal recessive multisystem disease which is caused by mutations in the CF transmembrane conductance receptor. Patients with CF have chronic respiratory infections which are the primary cause of morbidity and premature mortality (16). Patients with CF are infected with bacterial pathogens on an age-dependent timeline (16). Typically, Staphylococcus aureus and nonencapsulated Haemophilus influenzae are the first isolates from infants with CF (34, 35). However, Pseudomonas aeruginosa infections in children with CF are associated with progressive lung disease (30, 33). Microbiology is used for the diagnosis of P. aeruginosa, but successful P. aeruginosa isolation can be complicated in nonexpectorating populations of infants and young children with CF (4). The diagnosis and eradication of the initial P. aeruginosa infection with antibiotics to prevent chronic infection and mucoid transformation are important, since this diagnosis influences the quality of life and long-term patient survival (1, 2, 7, 11, 29, 30). Non-culture-based tests, like serology, should assist microbiology in the early diagnosis of P. aeruginosa infection.P. aeruginosa serology continues to be challenging without defined commercially available antigens licensed in the United States that reflect the molecular pathogenesis of P. aeruginosa upon adaptation to the host environment (13). Høiby (24) and Döring and Høiby (10) have detected an antibody response against a pool of antigens from common P. aeruginosa serotypes. Elevating antibody titers against this pool of antigens correlated with worsening P. aeruginosa infections and a poor clinical prognosis. The clinical progression of CF lung disease may be a reflection of the molecular pathogenesis of P. aeruginosa. Recent studies have correlated serological reactivity and known P. aeruginosa virulence factors. West et al. (42) showed that during the initial P. aeruginosa infection of children with CF, detection of serum antibodies to exotoxin A (ETA) and a P. aeruginosa lysate occurred earlier than detection of serum antibodies to elastase or alkaline phosphatase; subsequently, Corech et al. (6) detected antibodies to components of the type III secretion system (TTSS) at a time similar to that of P. aeruginosa Sup and earlier than ETA, showing the potential of measuring the antibody response to components of the TTSS as an indication of initial infection with P. aeruginosa in children with CF. This also indicated a role for TTSS in the initial P. aeruginosa pathogenesis of the CF lung.In the present study, a proteomic analysis was performed to obtain a global assessment of the host immune response during the initial P. aeruginosa infection of patients with CF. The goal was to identify a cellular component of P. aeruginosa that elicits an early immune response to P. aeruginosa infection to provide a stable immunogenic indication of P. aeruginosa infection relative to P. aeruginosa virulence factors that may fluctuate in expression during the course of P. aeruginosa infection, especially following transition from the acute to the chronic infection phase (41). Outer membrane protein L (OprL), a non-TTSS protein, was identified as an early immunogenic protein in the initial P. aeruginosa infection of patients with CF.     

An Engineered Human Antibody Fab Fragment Specific for Pseudomonas aeruginosa PcrV Antigen Has Potent Antibacterial Activity

Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute lung injury and mortality through the delivery of exotoxins by the type III secretion system (TTSS). PcrV is an important structural protein of the TTSS. An engineered human antibody Fab fragment that binds to the P. aeruginosa PcrV protein with high affinity has been identified and has potent in vitro neutralization activity against the TTSS. The instillation of a single dose of Fab into the lungs of mice provided protection against lethal pulmonary challenge of P. aeruginosa and led to a substantial reduction of viable bacterial counts in the lungs. These results demonstrate that blocking of the TTSS by a Fab lacking antibody Fc-mediated effector functions can be sufficient for the effective clearance of pulmonary P. aeruginosa infection.Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute and chronic infections in compromised individuals. It is a frequent causative agent of bacteremia in burn victims (32) and immunocompromised patients (18). It is also the most common cause of nosocomial gram-negative pneumonia (7, 25), especially in mechanically ventilated patients (25), and is the most prevalent pathogen in the lungs of individuals with cystic fibrosis (CF) (10, 17, 20). In CF, P. aeruginosa infection follows a well-established pattern of recurrent pulmonary infection in early childhood leading to the establishment of chronic infection in older CF patients, where it is a major contributing factor in the progressive decline in lung function and disease exacerbations leading to respiratory failure (10, 17).Morbidity and mortality associated with P. aeruginosa infections remain high despite the availability of antibiotics to which the bacterium is sensitive, and antibiotic resistance is an increasingly common problem in nosocomial infections (6).The type III secretion system (TTSS) is an important virulence determinant of P. aeruginosa in animal models of infection (15) and is required for the systemic spread of P. aeruginosa in a mouse pulmonary challenge model (31). The expression of a functional TTSS also correlates with poor prognosis in clinical infections (26, 27). This needle-like structure comprises a complex secretion and translocation machinery to inject a set of up to four different exotoxins (ExoS, ExoT, ExoU, and ExoY) directly into the cytoplasm of eukaryotic cells (9, 33, 34). Various strains of P. aeruginosa secrete different exotoxins. In addition, the TTSS can mediate direct cytotoxicity toward macrophages and neutrophils in the absence of exotoxins, a process called “oncosis,” requiring bacterial swarming in response to macrophage factors and leading to a direct perforation of the cell membranes (3, 4). In all of these functions of the TTSS, the needle tip protein, PcrV, is an essential component of the translocation apparatus.Antisera raised against PcrV in rabbits have been shown to block the translocation of Pseudomonas exotoxins into mammalian cells (12, 28) and to protect against lethality in a mouse model of acute pulmonary Pseudomonas infection (28, 29). Polyclonal anti-PcrV antibodies have also been shown to reduce lung damage and protect against bacteremia and septic shock in rat and rabbit pulmonary infection models (29) and to protect burned mice from infection (22). A mouse monoclonal anti-PcrV antibody, monoclonal antibody (MAb) 166, with potent neutralizing activity in mouse and rat models of Pseudomonas infection has also been described (11). This antibody inhibits the function of the TTSS in cell-based assays (11, 12). The MAb acts to prevent sepsis and mortality in an acute pulmonary infection model in mice when delivered either systemically or by intratracheal administration (11) and reduces lung damage due to Pseudomonas in a rat model (8). The antibody has activity when dosed either prophylactically or therapeutically in these models both as whole immunoglobulin G (IgG) and as a Fab fragment, indicating that the inhibition of TTSS function is sufficient to inhibit lung damage in pulmonary Pseudomonas infections.Here, we identify an engineered human antibody Fab fragment specific for the P. aeruginosa PcrV protein which competes with MAb 166 for binding to the same epitope on PcrV. The human Fab shows potent TTSS-neutralizing activity, equivalent to the activity of MAb 166, in cellular cytotoxicity assays. This Fab shows potent in vivo activity in protecting mice from potentially lethal doses of P. aeruginosa. In addition, the Fab mediates a substantial clearance of bacteria from the lungs of infected mice, suggesting that antagonism of the TTSS is sufficient not only to prevent damage to the pulmonary epithelium but also to restore normal immunological clearance mechanisms for the effective resolution of Pseudomonas infection in the complete absence of antibody effector functions.     

Flagellum-Mediated Biofilm Defense Mechanisms of Pseudomonas aeruginosa against Host-Derived Lactoferrin

Chronic infection with the gram-negative organism Pseudomonas aeruginosa is a leading cause of morbidity and mortality in human patients, despite high doses of antibiotics used to treat the various diseases this organism causes. These infections are chronic because P. aeruginosa readily forms biofilms, which are inherently resistant to antibiotics as well as the host''s immune system. Our laboratory has been investigating specific mutations in P. aeruginosa that regulate biofilm bacterial susceptibility to the host. To continue our investigation of the role of genetics in bacterial biofilm host resistance, we examined P. aeruginosa biofilms that lack the flgK gene. This mutant lacks flagella, which results in defects in early biofilm development (up to 36 h). For these experiments, the flgK-disrupted strain and the parental strain (PA14) were used in a modified version of the 96-well plate microtiter assay. Biofilms were challenged with freshly isolated human leukocytes for 4 to 6 h and viable bacteria enumerated by CFU. Subsequent to the challenge, both mononuclear cells (monocytes and lymphocytes) and neutrophils, along with tumor necrosis factor alpha (TNF-α), were required for optimal killing of the flgK biofilm bacteria. We identified a cytokine cross talk network between mononuclear cells and neutrophils that was essential to the production of lactoferrin and bacterial killing. Our data suggest that TNF-α is secreted from mononuclear cells, causing neutrophil activation, resulting in the secretion of bactericidal concentrations of lactoferrin. These results extend previous studies of the importance of lactoferrin in the innate immune defense against bacterial biofilms.Through the ability to form biofilms, bacteria change their susceptibility to antibiotics and the host''s immune system, contributing to chronic infections in humans. It''s estimated that biofilm infections cost $6.5 billion annually to treat (2). Moreover, most biofilm communities utilize genetically regulated processes to limit the effectiveness of a broad range of antimicrobial agents, likely resulting in even greater health care costs (54). Thus, the development of therapy that protects the host, or augments the clearance of biofilm infections, is vital to improved patient health and to reducing the cost burden of these infections. However, the immune response to biofilm infections is poorly characterized, and these mechanisms must be elucidated to direct the development of biofilm-specific therapies.Although much of the current understanding of host-pathogen interactions came from the study of planktonic bacteria, it is now clear that bacteria in the environment, including the human environment, live more often as communities of microorganisms (biofilms) than as single-cell suspensions (12, 44, 47). Bacteria within a biofilm differ from their planktonic counterparts in a number of ways, including an inherent resistance to antimicrobial agents and to clearance by the human immune system (23, 35, 36, 40, 44, 48, 54). We recently confirmed a direct link between Pseudomonas aeruginosa biofilm genetics and resistance to components of the human innate immune system (35). During this time, other papers have been published supporting our idea that P. aeruginosa and biofilms in general manipulate the immune system to their advantage (41, 55, 56). Most recently, O''Toole and colleagues demonstrated that P. aeruginosa biofilms growing on airway epithelial cells exhibited decreased virulence in the presence of the antibiotic tobramycin (1).The most classic example of Pseudomonas aeruginosa biofilm infections in humans are those associated with the lungs of cystic fibrosis (CF) patients, which correlate with increased morbidity and mortality (11, 13-15, 21, 22, 32, 33, 39, 53). Over three decades ago, Costerton and others demonstrated that lung material from CF patients contained P. aeruginosa likely in the form of biofilms (25, 33). More recently, Singh et al. showed that biofilms of P. aeruginosa existed in the CF lung (53). Collectively, these papers underscore the importance of biofilm formation in vivo and document the inability of the host''s immune system to clear the infections.Besides infections in the lungs of CF patients, P. aeruginosa also wreaks havoc in patients with corneal infections, burn wounds, implanted medical devices, and human immunodeficiency virus (46). Increased morbidity and mortality in these infections are linked to the ability of P. aeruginosa to form a biofilm (44, 45, 55). Schaber and colleagues (50) demonstrated the formation of biofilms on a mouse burn model after just 8 h postinoculation. In a 3-year study in Poland, P. aeruginosa was the most abundant organism isolated from burn wounds (5). Additionally, P. aeruginosa biofilm presence in chronic wounds is a likely culprit for the longevity of these infections by keeping the wound in a chronic state of inflammation (7, 31). These studies highlight the ubiquitous nature of this opportunistic pathogen and suggest a much larger role of P. aeruginosa biofilms in many chronic infections in humans.Alginate protects biofilm bacteria against macrophage phagocytosis (35). Besides the physical process of phagocytosis and the killing of bacteria by macrophages and neutrophils, a number of other antibacterial effector molecules are important in host defense. Singh and colleagues have shown that lactoferrin (LTF) prevents biofilm development (52). Over a decade ago, superoxide production by neutrophils was shown to be decreased in biofilm versus planktonic P. aeruginosa (26, 27). Other oxygen-dependent (nitric oxide [NO]) and oxygen-independent (lysozyme and myeloperoxidase [MPO]) responses are also reduced in magnitude or function in response to P. aeruginosa biofilms (9, 29, 30). These effector molecules are important in host defense, yet their effect on biofilm virulence or development, especially how they relate mechanistically, remains understudied. Nonetheless, it is now clear that genetic regulation within the biofilm bacteria plays a large role in resistance to the human host.In Salmonella enterica serovar Typhimurium and Escherichia coli, mutations in the flgK gene result in a nonmotile phenotype, as flgK is a vital gene involved in flagellar synthesis (24). This functional defect is also observed in P. aeruginosa. The lack of flagella in P. aeruginosa decreases the magnitude of early biofilm formation but does not prevent it (45). This defect is overcome with time as mutants lacking the flgK gene phenotypically approach wild-type biofilm levels after ∼36 h of growth. To determine if this genetic mutation affected biofilm susceptibility to components of the innate immune system, we grew biofilms of PA14 and isogenic flgK, challenged them with freshly isolated human leukocytes, and measured biofilm bacterial survival as previously described (35). In the absence of functional flagella, P. aeruginosa biofilm bacteria were killed by human leukocytes. To further dissect the interactions of the host cells with the biofilms, we developed a novel transwell biofilm killing assay that allowed for the separation of specific leukocyte populations (mononuclear cells from neutrophils) and the analysis of antimicrobial products such as superoxide, MPO, and LTF. Our data demonstrate that cytokine cross talk, as well as the production of components of the innate immune system, is important in generating mechanistic responses that in this case were able to kill biofilm bacteria that lacked intact flagella.     

Expression of B-Cell Activating Factor Enhances Protective Immunity of a Vaccine against Pseudomonas aeruginosa

B-cell activating factor (BAFF), a member of the TNF family, is a potent cytokine with stimulatory effects on B and T cells. To evaluate the potential of transient overexpression of BAFF to enhance vaccine immunogenicity, a replication-deficient adenovirus expressing full-length murine BAFF (AdBAFF) was tested in a mouse vaccine model against Pseudomonas aeruginosa. When coadministered with heat-killed P. aeruginosa, AdBAFF mediated a significant increase in anti-P. aeruginosa-specific serum and lung mucosal antibodies and resulted in improved protection against a lethal respiratory challenge with P. aeruginosa. This effect was independent of the site of administration of AdBAFF and was observed both when AdBAFF was given simultaneously with heat-killed P. aeruginosa as well as when AdBAFF was administered 4 weeks after immunization with heat-killed P. aeruginosa. These data demonstrate that a temporal increase in systemic BAFF levels is able to augment a P. aeruginosa-specific immune response upon immunization with heat-killed P. aeruginosa, suggesting that the immune-stimulatory effects of BAFF may be exploited as a molecular adjuvant for genetic vaccines.B-cell activating factor (BAFF, also called BLyS, Tall-1, or TNFSF13), a member of the TNF family, is an important cytokine that controls B-cell survival and maturation (3, 19, 27, 33, 43). BAFF is generated as a transmembrane molecule that is shed from the membrane upon furin cleavage (33, 43). Stromal cells in lymphoid tissue secrete BAFF, providing survival signals for developing B cells, and antigen-presenting cells secrete BAFF following activation (10, 25, 34). BAFF plays a crucial role in B-cell homeostasis, as demonstrated by the phenotypes of BAFF-deficient and BAFF-transgenic mice: BAFF-deficient mice have very few peripheral B cells due to a block in the final maturation stages of B cells in the spleen (12, 42), while BAFF transgenic mice have increased numbers of peripheral B cells, enlargement of the spleen, increased circulating immunoglobulin levels, and ultimately develop a systemic autoimmune disease, suggesting that a chronic excess of BAFF can break self tolerance (3, 13, 20, 28). In addition to its role in B-cell homeostasis, BAFF activates mature B cells in lymphoid tissues, induces immunoglobulin class switch, and delivers a strong costimulatory signal to T cells (5, 7, 16, 17, 25, 35, 36).Based on these observations, we hypothesized that transient overexpression of BAFF in vivo may enhance antigen-specific humoral immunity and may be useful for vaccines designed to induce antibodies against extracellular bacteria. With the knowledge that heat-inactivated Pseudomonas aeruginosa is an effective means to induce anti-P. aeruginosa immunity in mice, we used this as a clinically relevant model vaccine to study the ability of transient expression of BAFF to enhance immunogenicity (23). To achieve transient, high-level expression of BAFF in vivo, a replication-deficient adenovirus (Ad) vector was generated expressing the full-length murine BAFF cDNA (AdBAFF). Subcutaneous and intranasal administration of AdBAFF together with heat-killed P. aeruginosa resulted in significantly increased P. aeruginosa-specific serum and lung mucosal antibodies in C57BL/6 mice, and protection following a lethal pulmonary challenge with P. aeruginosa was observed in the AdBAFF-treated mice. Interestingly, protective anti-P. aeruginosa immunity was also improved when AdBAFF was administered 4 weeks after immunization with heat-killed P. aeruginosa, demonstrating that the transient overexpression of BAFF can augment an ongoing immune response following initial exposure to antigen. These results show that BAFF enhances antigen-specific humoral immunity and suggest that BAFF is a promising cytokine for the development of an improved vaccine against pathogens such as P. aeruginosa.     

Pseudomonas aeruginosa Alginate Promotes Burkholderia cenocepacia Persistence in Cystic Fibrosis Transmembrane Conductance Regulator Knockout Mice

Pseudomonas aeruginosa, a major respiratory pathogen in cystic fibrosis (CF) patients, facilitates infection by other opportunistic pathogens. Burkholderia cenocepacia, which normally infects adolescent patients, encounters alginate elaborated by mucoid P. aeruginosa. To determine whether P. aeruginosa alginate facilitates B. cenocepacia infection in mice, cystic fibrosis transmembrane conductance regulator knockout mice were infected with B. cenocepacia strain BC7 suspended in either phosphate-buffered saline (BC7/PBS) or P. aeruginosa alginate (BC7/alginate), and the pulmonary bacterial load and inflammation were monitored. Mice infected with BC7/PBS cleared all of the bacteria within 3 days, and inflammation was resolved by day 5. In contrast, mice infected with BC7/alginate showed persistence of bacteria and increased cytokine levels for up to 7 days. Histological examination of the lungs indicated that there was moderate to severe inflammation and pneumonic consolidation in isolated areas at 5 and 7 days postinfection in the BC7/alginate group. Further, alginate decreased phagocytosis of B. cenocepacia by professional phagocytes both in vivo and in vitro. P. aeruginosa alginate also reduced the proinflammatory responses of CF airway epithelial cells and alveolar macrophages to B. cenocepacia infection. The observed effects are specific to P. aeruginosa alginate, because enzymatically degraded alginate or other polyuronic acids did not facilitate bacterial persistence. These observations suggest that P. aeruginosa alginate may facilitate B. cenocepacia infection by interfering with host innate defense mechanisms.Respiratory failure due to lung infection is the major cause of mortality in cystic fibrosis (CF) patients. CF airways are colonized by more than one opportunistic bacterial pathogen, and Pseudomonas aeruginosa is a major pathogen. The other opportunistic bacterial pathogens that are frequently isolated from CF airways include Haemophilus influenzae, Staphylococcus aureus, the Burkholderia cepacia complex (BCC), Stenotrophomonas maltophilia, and methicillin-resistant S. aureus (7). Most individuals with CF experience a characteristic age-related pattern of pulmonary colonization and intermittent exacerbations involving H. influenzae and S. aureus, followed by P. aeruginosa (4, 5). Similarly, accumulating evidence suggests that P. aeruginosa can promote colonization by less commonly observed bacteria, such as S. maltophilia, Achromobacter xylosoxidans, and Mycobacterium abscessus (43). P. aeruginosa has also been implicated in promoting BCC pathogenesis by increasing the adherence of BCC to respiratory epithelial cells and upregulating the expression of BCC virulence factors (17, 31, 32).Chronic P. aeruginosa infections are often associated with a mucoid phenotype due to the production of large quantities of the acidic exopolysaccharide alginate (5). Alginate is an important extracellular virulence factor and has been shown to impair host innate defenses related to phagocytes (1, 13, 15, 18, 26, 30). In CF airways, P. aeruginosa is found in the airway lumen, and hence one may expect large amounts of alginate in airways along with host products. Sputum samples from CF patients have been shown to contain 50 to 200 μg/ml alginate (23, 30). In fact, it is likely that there are much higher concentrations of alginate in CF airways, as sputum samples are mixed with host secretions and hence the concentration of alginate may be underestimated. Since BCC infection generally occurs in patients who have been chronically colonized with mucoid P. aeruginosa, we hypothesized that alginate in the airways may prevent detection of BCC by phagocytes and facilitate colonization of CF lungs by BCC. To test this hypothesis, we infected gut-corrected CF mice with Burkholderia cenocepacia strain BC7 suspended in either phosphate-buffered saline (PBS) (BC7/PBS), P. aeruginosa alginate (BC7/alginate), or enzymatically degraded alginate (BC7/ED-alginate) and examined the persistence of bacteria and the associated lung inflammation. We also examined the effects of alginate on phagocytosis of B. cenocepacia by macrophages and neutrophils and the proinflammatory responses of airway epithelial cells to B. cenocepacia infection.     

EuroCareCF Quality Assessment of Diagnostic Microbiology of Cystic Fibrosis Isolates

The identification of microbial species from respiratory specimens and their susceptibility to antimicrobial agents are among the most important diagnostic measures of care for patients with cystic fibrosis (CF). Under the umbrella of EuroCareCF, two quality assurance trials of CF microbiology were performed in 2007 and 2008. Nine formulations with CF bacterial isolates were dispatched. A total of 31/37 laboratories from 18/21 European countries participated in the 2007 and 2008 trials. The common CF pathogens Pseudomonas aeruginosa and Staphylococcus aureus were correctly identified by almost all participants in both trials, even if the strains presented uncommon phenotypes. Burkholderia cenocepacia IIIB and Burkholderia vietnamensis CF isolates, however, were correctly assigned to the species level by only 26% and 27% of the laboratories, respectively. Emerging pathogens such as Achromobacter xylosoxidans, Inquilinus limosus, and Pandoraea pnomenusa were also not detected or were misclassified by many laboratories. One participant correctly identified all CF isolates in both trials. The percentages of correct classifications (susceptible, intermediate, resistant) by antimicrobial susceptibility testing ranged from 55 to 100% (median, 96%) per isolate and drug. The shortcomings in the diagnostics of rare and emerging pathogens point to the need for continuing education in CF microbiology and suggest the establishment of CF microbiology reference laboratories.The monogenic disorder cystic fibrosis (CF) predisposes individuals to chronic airway infections with opportunistic bacterial pathogens (16, 18, 19, 23). The bacteria most frequently isolated from the sputum of patients with CF by standard aerobic microbiological methods are Staphylococcus aureus, noncapsulated Haemophilus influenzae, and Pseudomonas aeruginosa (6). Individuals with CF are, moreover, susceptible to chronic respiratory tract infection with gram-negative bacterial species, which are intrinsically resistant to a broad range of antimicrobial agents and which are usually poor airway colonizers and not pathogenic for healthy persons (7). These rare and/or emerging pathogens in CF include Stenotrophomonas maltophilia (1); Achromobacter (Alcaligenes) xylosoxidans (15); Inquilinus limosus (20); and several species within the genera Burkholderia (12, 13), Ralstonia (12), and Pandoraea (11). Recent 16S rRNA gene profiling of CF respiratory secretions uncovered a further layer of complexity of CF microbiology (17). The bacterial community within the CF lung was found to be polymicrobial in nature and to include a range of anaerobic species primarily within the genera Prevotella, Veillonella, Propionibacterium, and Actinomyces (17, 21, 25).A further characteristic feature of CF isolates that impedes the straightforward identification of taxa is their broad spectrum of numerous and often atypical phenotypes (4, 9, 14, 22). For example, a P. aeruginosa clone may diversify in CF lungs into different morphotypes (14), such as small-colony variants, alginate-overproducing mucoid variants, nonpigmented variants, or colonies with visible autolysis or autoaggregative behavior, all of which carry other adaptive mutations, metabolic features, and antimicrobial susceptibility patterns.Present day CF microbiology services play a central role in the management of CF. Sensitive issues are the detection of transmissible pathogens, the emergence of multidrug-resistant variants, and the control of the efficacy of hygienic measures. To master these tasks, the clinical microbiology laboratory should have profound knowledge of the recent progress in the molecular taxonomy of CF pathogens, particularly among the betaproteobacteria, and the broad spectrum of uncommon phenotypes of isolates, particularly those from elderly CF patients (4). These demands are not trivial, and hence, the authors organized two quality assurance trials to address the issue of whether current knowledge in CF microbiology is translated into the microbiology services provided by the CF clinic. The trials asked for the species identification and antimicrobial susceptibilities of isolates from CF airways. Laboratories from 26 European countries which provide CF microbiology services for the largest CF centers in their home country were invited to participate. The trials identified shortcomings in the detection of rare and/or emerging pathogens which point to the need for continuing education in CF microbiology to promptly translate state-of-the-art knowledge into the daily practice of the clinical microbiology laboratory.     

Evaluation of Flagella and Flagellin of Pseudomonas aeruginosa as Vaccines

Pseudomonas aeruginosa is a serious pathogen in hospitalized, immunocompromised, and cystic fibrosis (CF) patients. P. aeruginosa is motile via a single polar flagellum made of polymerized flagellin proteins differentiated into two major serotypes: a and b. Antibodies to flagella delay onset of infection in CF patients, but whether immunity to polymeric flagella and that to monomeric flagellin are comparable has not been addressed, nor has the question of whether such antibodies might negatively impact Toll-like receptor 5 (TLR5) activation, an important component of innate immunity to P. aeruginosa. We compared immunization with flagella and that with flagellin for in vitro effects on motility, opsonic killing, and protective efficacy using a mouse pneumonia model. Antibodies to flagella were superior to antibodies to flagellin at inhibiting motility, promoting opsonic killing, and mediating protection against P. aeruginosa pneumonia in mice. Protection against the flagellar type strains PAK and PA01 was maximal, but it was only marginal against motile clinical isolates from flagellum-immunized CF patients who nonetheless became colonized with P. aeruginosa. Purified flagellin was a more potent activator of TLR5 than were flagella and also elicited higher TLR5-neutralizing antibodies than did immunization with flagella. Antibody to type a but not type b flagella or flagellin inhibited TLR5 activation by whole bacterial cells. Overall, intact flagella appear to be superior for generating immunity to P. aeruginosa, and flagellin monomers might induce antibodies capable of neutralizing innate immunity due to TLR5 activation, but solid immunity to P. aeruginosa based on flagellar antigens may require additional components beyond type a and type b proteins from prototype strains.Pseudomonas aeruginosa is an opportunistic pathogen responsible for a large proportion of ventilator-associated, hospital acquired pneumonia and is also a major cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa is motile via a single polar flagellum that has the added structural feature of being glycosylated (39). Flagellin is the primary protein component of the flagellar filament, and it can be classified into two serotypes, types a and b. Flagella carry out many functions, such as motility and attachment of bacteria to host cells, and can also elicit the activation of the host inflammatory response via Toll-like receptor 5 (TLR5) (6, 15, 29, 31). Importantly, promising results in terms of prevention of the acquisition of P. aeruginosa infection in CF patients immunized with a bivalent type a and b flagellum vaccine have been published (12).Several animal studies have not only demonstrated the importance of flagella as a virulence factor in P. aeruginosa but also validated them, or their flagellin component, as target antigens for vaccination. In the burned-mouse model of infection, chemically mutagenized or genetically produced flagellum-negative strains were less virulent than flagellum-positive strains (5, 26). It has also been shown with this model that motility is necessary for dissemination from the site of infection, since an intact flagellum structure is essential for death due to sepsis (5). In a neonatal model of acute P. aeruginosa pulmonary infection, flagella were essential for full virulence (14), although this was not found to be the case for adult mice with pulmonary P. aeruginosa infection (6). In regard to protection mediated by flagella or flagellin, immunization with flagella provided protection against infection and decreased the spread to major organs in the burned-mouse model (19). In a rat model of P. aeruginosa-induced pneumonia, administration of human monoclonal antibodies (MAbs) to flagella provided protection against infection and decreased lung injury (24), and another set of human MAbs provided protection in a murine model of pneumonia during neutropenia (28). A DNA vaccine encoding recombinant type a or type b P. aeruginosa flagellin also induced protective immunity against lethal P. aeruginosa lung infection (33), although this study curiously found better heterologous protection than homologous protection when DNA encoding wild-type flagellin was incorporated into the vaccine. A fusion protein of outer membrane protein F (OprF) residues 311 to 341, mature OprI residues 21 to 83, and flagellins a and b (termed OprF311-341-OprI-flagellins) generated significant immune responses in mice and promoted enhanced clearance of strain PA01 in a pulmonary challenge model (42). None of these studies directly compared the vaccine potential of flagellin with that of flagella.In addition to being highly immunogenic, the flagellin component of flagella serves as a pathogen-associated molecular pattern (PAMP), activating TLR5 and inducing innate immunity in the lung, stimulating a protective inflammatory response that contributes to the eradication of the pathogen (15, 32, 33, 35). Instillation of recombinant flagellin into the lungs of mice elicits a significant induction of innate immunity (20), and application of flagellin to the cornea of mice or intraperitoneal (i.p.) injection prior to corneal injury and local P. aeruginosa infection protects against pathological destruction of this tissue (22, 23). Finally, overexpression of flagellin monomers enhances virulence of P. aeruginosa (6).Of great interest is that the TLR5-binding domain of flagellin is not exposed in the intact flagella (36), and thus, flagellin monomers must be released or extracted from the intact flagella to promote TLR5 activation. Therefore, the comparative TLR5 agonist activity of flagellin, flagella, and even intact P. aeruginosa bacteria has not been evaluated, nor is it clear if the TLR5 activation component of flagellin would be immunogenic when immunizing with the intact polymeric flagella.Since P. aeruginosa serotype a and b flagella are conserved, contribute to virulence, stimulate innate immunity, and have induced protective efficacy in both animal (19, 24) and human (12) vaccine studies, it is clear that the flagellum or the flagellin monomer may be a useful target as a vaccine component, particularly as a carrier protein to link to protective carbohydrate antigens such as lipopolysaccharide (LPS) O-side chains or the alginate capsule (11, 30, 37). To our knowledge, no comparative analysis of the vaccine efficacy of flagellin versus that of flagella has been described for P. aeruginosa or other pathogens. Thus, it is not clear if it is flagellum or flagellin that is the best vaccine candidate, if either or both could be effectively utilized as a component of a conjugate vaccine, and if use of these vaccines could induce a state of enhanced susceptibility to infection by blocking flagellin-TLR5 interactions that promote effective innate immunity, as was found with antibodies induced by a DNA vaccine encoding P. aeruginosa flagellin (33). The purpose of this study was to compare whether immunity to P. aeruginosa flagella and that to flagellin are comparable or distinct and to evaluate if antibodies neutralizing TLR5 activation are induced and whether this impacted TLR5 activation by flagellin, flagella, or intact P. aeruginosa cells.     

Virulence and Cellular Interactions of Burkholderia multivorans in Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) patients are susceptible to life-threatening infections by the Burkholderia cepacia complex. We used leukocytes from CGD and healthy donors and compared cell association, invasion, and cytokine induction by Burkholderia multivorans strains. A CGD isolate, CGD1, showed higher cell association than that of an environmental isolate, Env1, which correlated with cell entry. All B. multivorans strains associated significantly more with cells from CGD patients than with those from healthy donors. Similar findings were observed with another CGD pathogen, Serratia marcescens, but not with Escherichia coli. In a mouse model of CGD, strain CGD1 was virulent while Env1 was avirulent. B. multivorans organisms were found in the spleens of CGD1-infected mice at levels that were 1,000 times higher than those found in Env1-infected mice, which was coincident with higher levels of the proinflammatory cytokine interleukin-1β. Taken together, these results may shed light on the unique susceptibility of CGD patients to specific pathogens.Chronic granulomatous disease (CGD) is a rare primary immunodeficiency resulting from genetic defects in the phagocyte NAPDH oxidase. It is characterized by life-threatening infections caused by specific bacteria and fungi, leading to pneumonias, tissue abscesses, and exuberant granuloma formation (38). The Burkholderia cepacia complex (Bcc) includes at least 10 distinct species and is a leading cause of bacterial infections in CGD (44). Patients with cystic fibrosis (CF) also develop Bcc infections with various outcomes, ranging from no change in clinical course to a more rapid deterioration of lung function to the dreadful cepacia syndrome, which is characterized by necrotizing pneumonia and sepsis (25, 45). Interestingly, Bcc rarely causes infection in healthy individuals, but it can infect patients undergoing bronchoscopies and other procedures (4).Within the Bcc, Burkholderia cenocepacia and Burkholderia multivorans are commonly isolated from CF and non-CF patients (4, 32); the rate of B. multivorans infection now exceeds that of B. cenocepacia at several CF centers (15). In contrast to the high transmissibility of some CF B. cenocepacia strains (i.e., the epidemic lineage ET12) (24, 25), CF B. multivorans infections likely reflect independent acquisitions from unrelated sources (24). Curiously, unlike B. cenocepacia, B. multivorans has been recovered from environmental samples only rarely (1, 24), and it is the most frequently found species among CGD patients (16, 17).The mechanisms by which the Bcc causes disease specifically in CF are not known. Bcc isolates can survive within macrophages (28, 33) and respiratory epithelial cells (5, 21) and can invade epithelial cells in vivo (8, 10) and persist in the lung (9, 10). Cell infection assays using monocytes, macrophages, and epithelial cells (10, 11, 29, 46) show great variability among individual Bcc strains, with no clear correlation between those isolated from CF patients and those isolated from the environment (22). For the most part, these studies have been carried out using tissue culture models (28, 29, 43) and, in some cases, CF human or CF mouse cell systems (34, 35).Much less is known about the interaction between the Bcc and CGD despite the availability of animal models for the disease (20, 31). B. cenocepacia induced the necrosis of human CGD neutrophils but not normal controls (6). Similarly to healthy people, normal mice are resistant to the Bcc and usually show only transient infections upon inoculation (8, 37). On the other hand, CGD mice are highly susceptible to Bcc infection and show clinical signs that are similar to those of the human disease (20, 31, 37).To address why B. multivorans is a pathogen in CGD, we initiated studies with strains isolated from CGD patients and CGD cells. Strains of B. multivorans differed in cell association and cell entry. We found a preferential association of bacteria with CGD instead of normal leukocytes as shown by microscopy and culture techniques. This preferential association is shared by another CGD pathogen, Serratia marcescens, but not by Escherichia coli. Finally, we demonstrate dramatic differences in virulence in B. multivorans strains in a mouse model of CGD.     

Impact of Pseudomonas aeruginosa Genomic Instability on the Application of Typing Methods for Chronic Cystic Fibrosis Infections

The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients in the United Kingdom and has emerged recently in North America. In this study, we report the analysis of 24 “anomalous” CF isolates of P. aeruginosa that produced inconsistent results with regard to either pulsed-field gel electrophoresis (PFGE) or PCR tests for the LES. We used a new typing method, the ArrayTube genotyping system, to determine that of the 24 anomalous isolates tested, 13 were confirmed as the LES. LES isolates could not be clearly distinguished from non-LES isolates by two other commonly used genetic fingerprinting tests, randomly amplified polymorphic DNA (RAPD) analysis and BOX-PCR, and varied considerably in their carriage of LES genomic islands and prophages. The genomic instability of the LES suggests that identification of this emerging transmissible strain could be a challenging task, and it questions whether discrimination is always a desirable feature of bacterial typing methods in the context of chronic CF infections.Chronic Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients are associated with a decline in lung function and increased mortality (11). Infection usually occurs during adolescence and, once established, the pathogen is impossible to eradicate (17). In recent years, the view of P. aeruginosa infection in CF patients has been altered because of the emergence of transmissible strains. Originally, it was thought that patients acquired their own unique strains from the environment and only in specific circumstances, such as siblings with CF, were patients found to share the same strain (35). However, in 1996 Cheng et al. (6) described the use of molecular typing to demonstrate the spread of a drug-resistant strain of P. aeruginosa (named the Liverpool epidemic strain [LES]) among patients in a children''s CF center in Liverpool, United Kingdom (6). Seven years later, an analysis of patient samples taken after the year 2000 identified the LES in 79% of 80 P. aeruginosa-colonized CF patients in the Liverpool adult CF center, confirming the spread and longevity of LES infections (29). In a survey of 31 CF centers in England and Wales in 2004, involving more than 1,200 isolates of P. aeruginosa, the LES was identified as the most common clone (33). Furthermore, the LES has also been identified in Scotland (10). Recently, cases of CF patients infected with the LES have also emerged in Canada (1).In addition to its transmissibility, the LES appears to be more aggressive than other P. aeruginosa strains. It has been shown to replace previously established strains of P. aeruginosa (superinfection) (24), has infected both non-CF parents of a CF patient (25), and has infected a pet cat, causing significant morbidity (26). Furthermore, the LES is associated with greater morbidity (2) and increased renal failure (3) and appears to have enhanced survival in air (30).Patient segregation based on LES status requires simple but effective strain typing methods with adequate discriminatory powers to achieve both the initial identification of the LES and subsequent epidemiological surveillance. The genome of P. aeruginosa is a mosaic structure consisting of a core genome and a variable accessory genome (23). The accessory genome includes large insertions, such as prophages and genomic islands, contributing to genome size variations between 5.2 and 7 Mb (38). The earliest archived isolate of the LES (LESB58, from 1988) has recently been genome sequenced (40), revealing the presence of six prophages and five genomic islands.Specific PCR assays have been developed for detection of the LES (31, 34). Following reports of false positives for the original marker PS21 (20), a second marker (LES-F9) was identified (34). Neither PS21 nor LES-F9, which map to separate genomic islands (LESGI-3 and LESGI-1, respectively), is 100% specific to the LES, but the combination of the two has not been reported previously in any non-LES strain. The use of PCR assays in routine diagnostic tests in Liverpool and elsewhere in the United Kingdom has led to the implementation of successful segregation measures. However, we have a number of putative LES CF isolates that do not give concordant results with respect to the presence of PS21, the presence of LES-F9, and the molecular typing method pulsed-field gel electrophoresis (PFGE). The Clondiag ArrayTube (AT) system is a portable method for interrogating both the conserved and accessory genomes of P. aeruginosa isolates. The process is rapid, relatively inexpensive, and robust and has been used to type P. aeruginosa isolates from diverse habitats (39).Here, we report the use of the AT system to resolve the strain status of a collection of suspected LES isolates of P. aeruginosa from CF patients. We provide evidence that the genomic instability exhibited by the LES could impact the validity of routine typing schemes, causing both false-positive and false-negative identification, and suggest that normal paradigms for bacterial typing may be limited in the context of chronic infections in CF.     

Pseudomonas aeruginosa Genotype Prevalence in Dutch Cystic Fibrosis Patients and Age Dependency of Colonization by Various P. aeruginosa Sequence Types

The patient-to-patient transmission of highly prevalent Pseudomonas aeruginosa clones which are associated with enhanced disease progression has led to strict segregation policies for cystic fibrosis (CF) patients in many countries. However, little is known about the population structure of P. aeruginosa among CF patients. The aim of the present cross-sectional study was to determine the prevalence and genetic relatedness of P. aeruginosa isolates from CF patients who visited two major CF centers in The Netherlands in 2007 and 2008. These patients represented 45% of the Dutch CF population. P. aeruginosa carriage in the respiratory tract was determined by standard microbiological culture techniques, and all phenotypically different isolates in the first specimens recovered in 2007 and 2008 were genotyped by multilocus sequence typing. A total of 313 (57%) of 551 patients whose samples were cultured carried P. aeruginosa. Two sequence types (STs), ST406 and ST497, were found in 15% and 5% of the patients, respectively, and 60% of the patients harbored a strain that was also found in at least two other patients. The risk ratios for carrying ST406 and ST497 were 17.8 (95% confidence interval [CI], 7.2 to 43.6) for those aged between 15 and 24 years and 6 (95% CI, 1.4 to 26.1) for those aged >25 years. ST406 and ST497 were not genetically linked to previously described epidemic clones, which were also not found in this CF population. The population structure of P. aeruginosa in Dutch CF patients is characterized by the presence of two prevalent STs that are associated with certain age groups and that are not genetically linked to previously described epidemic clones.Pseudomonas aeruginosa is a ubiquitous, versatile bacterium that can infect humans as well as plants and animals. The species is infamous for causing nosocomial infections in immunocompromised patients and patients in intensive care units and is a major cause of morbidity and mortality in patients with cystic fibrosis (CF) (26).The widely held belief that CF patients acquire P. aeruginosa strains mainly from their inanimate environment, with most patients being colonized by unique strains, has been challenged by reports indicating that P. aeruginosa clones may frequently be transmitted between CF patients (3, 6, 18, 19, 23, 24). Some of these clones, such as the Liverpool epidemic strain and the Melbourne epidemic strain, have been associated with enhanced disease progression and higher rates of mortality, respectively (1, 13). In The Netherlands, the patient-to-patient transmission of P. aeruginosa was documented during a summer camp (4). These findings have led to strict segregation policies for CF patients in many countries, including The Netherlands. However, despite these studies, there is little information on the population structure of P. aeruginosa within populations of CF patients. We therefore investigated the prevalence and genetic relatedness of P. aeruginosa isolates compared to those of the international known genotypes in an unbiased cohort representing 45% of the CF population in The Netherlands in 2007 and 2008.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Nonrandom Distribution of Pseudomonas aeruginosa and Staphylococcus aureus in Chronic Wounds

The spatial organization of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds was investigated in the present study. Wound biopsy specimens were obtained from patients diagnosed as having chronic venous leg ulcers, and bacterial aggregates in these wounds were detected and located by the use of peptide nucleic acid-based fluorescence in situ hybridization and confocal laser scanning microscopy (CLSM). We acquired CLSM images of multiple regions in multiple sections cut from five wounds containing P. aeruginosa and five wounds containing S. aureus and measured the distance of the bacterial aggregates to the wound surface. The distance of the P. aeruginosa aggregates to the wound surface was significantly greater than that of the S. aureus aggregates, suggesting that the distribution of the bacteria in the chronic wounds was nonrandom. The results are discussed in relation to our recent finding that swab culturing techniques may underestimate the presence of P. aeruginosa in chronic wounds and in relation to the hypothesis that P. aeruginosa bacteria located in the deeper regions of chronic wounds may play an important role in keeping the wounds arrested in a stage dominated by inflammatory processes.Chronic wounds, such as diabetic foot ulcers, pressure ulcers, and venous leg ulcers, are an increasing problem worldwide. One to 2% of the population in developed countries develops chronic wounds, a condition associated with severe patient suffering, the loss of employment, a reduced quality of life, and high costs to the health care system (13). Detailed knowledge about chronic wounds is required in order to develop better wound treatment and management strategies.A normal wound healing process involves four main phases: (i) coagulation, (ii) inflammation, (iii) cell proliferation and repair of the matrix, and (iv) epithelialization and remodeling of the scar tissue (23). However, chronic wounds are believed to be captured in the inflammatory phase, where persistent influx and elevated activity of polymorphonuclear neutrophils (PMNs) occur (1). Although PMNs play a critical role in the host defense and wound healing, they release cytolytic enzymes, free oxygen radicals, inflammatory mediators, and matrix metalloproteases, which cause local tissue damage in the host (22, 23, 26).It is known that the microflora of chronic wounds comprises multiple species. In a bacterial profiling study, Gjødsbol et al. found that chronic venous leg ulcers harbored Staphylococcus aureus (in 93.5% of the ulcers), Enterococcus faecalis (71.7%), Pseudomonas aeruginosa (52.2%), coagulase-negative staphylococci (45.7%), Proteus species (41.3%), and anaerobic bacteria (39.1%) (12). S. aureus and P. aeruginosa are opportunistic pathogenic bacteria and are widely known to cause chronic biofilm-based infections in their hosts. S. aureus is most commonly isolated from chronic wounds (8, 12, 15, 17) and, in certain situations, can express a number of potential virulence factors and surface proteins which promote its adherence to the damaged tissue and decrease neutrophil functions and immune responses of the host (10, 11). P. aeruginosa often causes biofilm-based chronic infections and expresses virulence factors, in particular, rhamnolipid, that can eliminate the activity of PMNs (4, 16). A number of studies have demonstrated that P. aeruginosa is frequently present in chronic wounds (12, 17) and have provided evidence that the bacteria are located in aggregates enclosed in extracellular polymeric matrix material as found in biofilms (17). Furthermore, chronic wounds that harbored P. aeruginosa were larger than those that did not, and the healing process also seemed to be more severely hindered for those wounds (12, 14, 20).Biofilms are bacterial aggregates enclosed in a self-produced extracellular polymeric matrix (6, 21, 25). In clinical environments biofilms can form on dead or living tissues, mucosal surfaces, or the surfaces of medical devices in the host. The bacteria in biofilms often display characteristics different from those of their planktonic counterparts, such as increased resistance to the activities of the host immune system and tolerance to antimicrobial treatments (7). Such characteristics are important, since biofilms are involved in many chronic bacterial infections. Recent studies have shown the presence of bacterial biofilms in chronic wounds (9, 15, 17). Although the role of biofilms in chronic wounds is not yet fully understood, it is believed that their existence may be one of the reasons for impaired wound healing (4, 16).We previously demonstrated that there is a lack of correlation between the bacteria detected by standard culturing and those detected directly by peptide nucleic acid (PNA)-based fluorescence in situ hybridization (FISH) in chronic wound samples (17). While S. aureus was detected more frequently by swab sample cultivation than by PNA-FISH, the opposite was true for P. aeruginosa. This lack of correlation between detection by swab sample cultivation and PNA-FISH may be due to the ability of the different bacterial species to colonize different regions of chronic wounds. Swab sample cultivation identifies the microorganisms present in the surface region of the wound but may not detect microorganisms located inside the wound bed. Accordingly, in the present report, we present evidence that S. aureus primarily colonizes the region of chronic wounds which is close to the surface, whereas P. aeruginosa primarily colonizes the deeper regions of chronic wounds. The ability of P. aeruginosa to colonize the deeper regions of chronic wounds may be due to the ability of this organism to produce virulence factors which destroy PMNs (4, 16), and it may play an important role in keeping the wounds arrested in a stage dominated by inflammatory processes.     

The Type 2 Secretion Pseudopilin,gspJ, Is Required for Multihost Pathogenicity of Burkholderia cenocepacia AU1054

Burkholderia cenocepacia AU1054 is an opportunistic pathogen isolated from the blood of a person with cystic fibrosis. AU1054 is a multihost pathogen causing rapid pathogenicity to Caenorhabditis elegans nematodes. Within 24 h, AU1054 causes greater than 50% mortality, reduced growth, emaciated body, distended intestinal lumen, rectal swelling, and prolific infection of the nematode intestine. To determine virulence mechanisms, 3,000 transposon mutants were screened for attenuated virulence in nematodes. Fourteen virulence-attenuated mutants were isolated, and the mutant genes were identified. These genes included paaA, previously identified as being required for full virulence of B. cenocepacia K56-2. Six mutants were restored in virulence by complementation with their respective wild-type gene. One of these contained an insertion in gspJ, predicted to encode a pseudopilin component of the type 2 secretion system (T2SS). Nematodes infected with AU1054 gspJ had fewer bacteria present in the intestine than those infected with the wild type but still showed rectal swelling. The gspJ mutant was also defective in pathogenicity to onion and in degradation of polygalacturonic acid and casein. This result differs from previous studies where no or little role was found for T2SS in Burkholderia virulence, although virulence factors such as zinc metalloproteases and polygalacturonase are known to be secreted by the T2SS. This study highlights strain specific differences in B. cenocepacia virulence mechanisms important for understanding what enables environmental microbes to function as opportunistic pathogens.The betaproteobacterium Burkholderia cenocepacia is a member of the Burkholderia cepacia complex (BCC), now consisting of 17 classified species (72). Members of the BCC are ubiquitous in the environment, metabolically diverse, and beneficial or pathogenic to a variety of organisms and have large and dynamic multireplicon genomes (11, 48). Originally described as a pathogen to onion plants (9), members of the BCC have emerged as opportunistic pathogens of serious concern to persons with cystic fibrosis (CF) or chronic granulomatous diseases (26, 29, 36). Although Pseudomonas aeruginosa is more commonly isolated from CF infections, infections with BCC also occur and are a serious concern because of their inherent multidrug resistance and correlation with the severe loss of lung function, sepsis, and fatality referred to as cepacia syndrome (36). Infections of CF patients arise from patient-to-patient transmission of epidemic clones or sporadically from a presumed environmental source (29). Epidemic strains of B. cenocepacia and B. multivorans are correlated with the incidence of cepacia syndrome (49, 75). The PHDC epidemic clone strain, B. cenocepacia AU1054 (called AU1054 hereafter), is a multihost pathogen with high virulence to the nematode Caenorhabditis elegans and to onions (13, 44, 64).The C. elegans model for the study of bacterial pathogenicity was originally developed for P. aeruginosa (20, 69), which causes nematode mortality by two mechanisms termed fast killing (within hours, toxin mediated) (46, 69) and slow killing (in days, infection mediated) (69). Virulence factors required for C. elegans killing are sometimes also involved in multihost virulence (e.g., plant and animal) (45). The C. elegans model has now been established for the study of virulence of a large variety of pathogens including BCC (10, 19, 41). AU1054 is highly pathogenic to C. elegans killing nematodes fast on minimal medium where P. aeruginosa kills slowly by an unknown mechanism (64).Type 2 secretion systems (T2SSs) are required for the secretion of many toxins and enzymes that contribute to virulence, notably exotoxin A and cholera toxin produced by P. aeruginosa and Vibrio cholerae, respectively (23, 62). Previous studies did not detect a significant defect in virulence of B. pseudomallei, B. cenocepacia, and B. vietnamiensis strains mutant in T2SS, although the T2SS is required for B. gladioli pathogenicity to mushrooms (14, 21, 24, 41). However, expression of the T2SS genes was found to be induced in synthetic CF sputum medium relative to soil extract by strains AU1054 and J2315; although the latter was unexpectedly found to have a 110-bp deletion in gspL, known to be required for T2SS function (34, 73, 74). These contradictory results suggest that it is possible that the T2SS is important for BCC to function as opportunistic pathogens.We describe here the application of the C. elegans model for the study of multihost pathogenicity of the CF epidemic B. cenocepacia strain AU1054. Our goal was to identify genes required for AU1054 pathogenicity using the C. elegans model and to determine the role of these genes in pathogenicity to multiple hosts.     

Translocation of Pseudomonas aeruginosa from the Intestinal Tract Is Mediated by the Binding of ExoS to an Na,K-ATPase Regulator,FXYD3

The intestinal tract is considered the most important reservoir of Pseudomonas aeruginosa in intensive care units (ICUs). Gut colonization by P. aeruginosa underlies the development of invasive infections such as gut-derived sepsis. Intestinal colonization by P. aeruginosa is associated with higher ICU mortality rates. The translocation of endogenous P. aeruginosa from the colonized intestinal tract is an important pathogenic phenomenon. Here we identify bacterial and host proteins associated with bacterial penetration through the intestinal epithelial barrier. We first show by comparative genomic hybridization analysis that the exoS gene, encoding the type III effector protein, ExoS, was specifically detected in a clinical isolate that showed higher virulence in silkworms following midgut injection. We further show using a silkworm oral infection model that exoS is required both for virulence and for bacterial translocation from the midgut to the hemolymph. Using a bacterial two-hybrid screen, we show that the mammalian factor FXYD3, which colocalizes with and regulates the function of Na,K-ATPase, directly binds ExoS. A pulldown assay revealed that ExoS binds to the transmembrane domain of FXYD3, which also interacts with Na,K-ATPase. Na,K-ATPase controls the structure and barrier function of tight junctions in epithelial cells. Collectively, our results suggest that ExoS facilitates P. aeruginosa penetration through the intestinal epithelial barrier by binding to FXYD3 and thereby impairing the defense function of tight junctions against bacterial penetration.Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of infection-related mortality among individuals with compromised immune systems. Fatality rates among patients infected with P. aeruginosa are higher than those among patients infected with any other opportunistic Gram-negative bacterium (48, 51). The lungs are a major site of P. aeruginosa infection in ill patients; however, a considerable number of such infections occur through direct contamination of the lungs by gastrointestinal flora or through hematogenous spread from the intestine to the lungs (51). In particular, the presence of highly virulent strains of P. aeruginosa within the intestinal tract alone is the main source of sepsis and death among immunocompromised patients, even in the absence of established extraintestinal infection and bacteremia (34, 41, 51). Furthermore, the lethal effects of intestinal P. aeruginosa are dependent upon its ability to adhere to and disrupt the intestinal epithelial barrier (1).The intestinal tract is considered to be the most important reservoir of P. aeruginosa (2). The rate of mortality of patients in intensive care units (ICUs) suffering from intestinal colonization by P. aeruginosa is significantly higher than that of patients without such colonization (34). The notion that gut colonization by P. aeruginosa sets the stage for the underlying development of invasive infection is supported by reports demonstrating a reduction in rates of ICU-acquired infection owing to a decontamination of the digestive tract (5, 31, 46). Recently, the importance of intestinal P. aeruginosa as a cause of mortality in critically ill patients was demonstrated in a randomized prospective study (11). Patients were subjected to selective antibiotic decontamination of the digestive tract through the oral administration of nonabsorbable antibiotics. This resulted in decreased mortality and was associated with a decrease in fecal P. aeruginosa. The translocation of P. aeruginosa from the colonized intestinal tract, a process whereby endogenous intestinal P. aeruginosa relocates extraluminally, is considered an important pathogenic phenomenon.The importance of the translocation of P. aeruginosa from the colonized intestinal tract in causing gut-derived septicemia was determined by exploiting leukopenic mice (35, 38, 48). In addition, most clinical blood isolates, but not human respiratory isolates, have been shown to cause lethal endogenous bacteremia in leukopenic mice (15, 24). Cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1α have been implicated in the translocation of P. aeruginosa in gut-derived sepsis of leukopenic mice (36, 37). Furthermore, multidrug efflux systems of P. aeruginosa, such as mexAB-oprM, have been reported to play a key role not only in the invasiveness of the bacteria into or transmigration of the bacterium across Madin-Darby canine kidney (MDCK) cells but also in the ability of the bacteria to kill leukopenic mice (24). However, it is still unclear at the molecular level how P. aeruginosa exploits multidrug efflux systems to penetrate the epithelial cell barrier.P. aeruginosa uses a type III secretion apparatus to inject effectors into host cells. The P. aeruginosa type III secretion system seems to have fewer effectors than any other bacterial type III secretion system: only four effector proteins, ExoS, ExoT, ExoU, and ExoY, of the P. aeruginosa type III secretion system have been identified. ExoS is a bifunctional toxin possessing an N-terminal Rho GTPase-activating protein (RhoGAP) activity that targets small GTPases (18, 20, 22, 49) and a highly promiscuous C-terminally encoded ADP-ribosylation activity (ADPRT) toward small GTP-binding proteins (8, 9, 20, 23). The RhoGAP activity of ExoS causes the disruption of the host cell actin cytoskeleton through modulating the switch between an active GTP-bound form and an inactive GDP-bound form. The ADPRT activity of ExoS has several effects on the host cell, such as a disruption of the actin cytoskeleton, inhibition of DNA synthesis, and cell death. The eukaryotic cofactor 14-3-3 is required for the activation of the ADPRT activity of ExoS (20). ExoT is closely related to ExoS and is also a bifunctional toxin with N-terminal GAP activity and C-terminal ADPRT activity (20). ExoU is a potent necrotizing toxin with phospholipase activity that is able to cause rapid cell death in eukaryotic cells (20). ExoY is an adenylate cyclase, and the injection of ExoY into mammalian cells causes an elevation of intracellular cyclic AMP (cAMP) levels, although the significance of ExoY in infection remains unclear (20). Recently, it was reported that infection of polarized airway epithelial cells with P. aeruginosa expressing type III effectors (ExoS, ExoT, and ExoY) disrupts intact tight junctions (TJs) by altering the distribution of the TJ proteins ZO-1 and occludin (44). However, the precise molecular mechanism by which the type III effector induces the disruption of TJs remains unknown. Although many binding partners of the P. aeruginosa type III effectors have been reported (20), it remains unclear how P. aeruginosa penetrates the epithelial cell barrier on the basis of interactions between type III effectors and such substrate proteins. Hauser previously suggested that the disruption of the cytoskeletal structure mediated by ExoS may contribute to a reduction in cell-cell adherence, which in turn may facilitate P. aeruginosa penetration through epithelial barriers, but there is no direct experimental confirmation of this hypothesis at present (20).Our aim here was to reveal the precise molecular mechanism by which P. aeruginosa disrupts TJs and penetrates the epithelial cell barrier.